The field of the invention relates to therapeutics and diagnostics that make use of macromolecules as delivery agents.
The use or hypothetical use of certain macromolecules as delivery agents to target appended drugs and diagnostic agents is known, but currently not without severe limitations. Attempts with therapeutics have yielded only limited clinical success due to a lack of inexpensive and nontoxic molecular backbones to which drugs and target substrates of sufficient load can be attached. Similar problems plague current cardiovascular and tumor imaging techniques, which make use of appended agents for magnetic resonance imaging (MRI) and computed tomography (CT).
Focus has thus centered on the delivery agent""s molecular backbone, which function is to carry drugs, substrates, and imaging and other diagnostic molecules for delivery to specific cell tissues. The most commonly employed backbones to date are dextran, polylysine, synthetic copolymers, starburst dendrimers, and human serum albumin.
Dextran, a branched polymer of glucose, has an extensive human-use experience, and offers the highest ratio of attachment sites per molecular weight. It is also very hydrophilic, which permits a low injection volume. While dextran is relatively inexpensive, it has the disadvantage of having insufficient chemical flexibility in its usual attachment sites and a high incidence of unwanted cross-linking that results from standard means of attachment.
Polylysine, by contrast, is extremely expensive and has a very limited human-use experience. Despite this, polylysine has the advantage of being available in various chain lengths and the further advantage of its side chains being readily amenable to chemical attachment of drugs and receptor substrates. Consequently, polylysine is frequently employed to test prospective drug delivery systems in animals.
Synthetic copolymers, e.g., as described by Krinick et al., Makromol. Chem. 191:839-856 (1990), offer linearity and a net neutral charge which increases diffusion. Synthetic copolymers offer further advantage in that they may be synthesized in bulk. However, their synthesis requires multiple steps that, if not complex, are nevertheless time consuming, expensive, and therefore inefficient. Further, there is limited human-use experience with synthetic copolymer backgrounds.
Starburst dendrimers (see Tomalia et al. (1985) Polymer J. 17:117-132) are advantageous in that they provide less molecular weight heterogeneity, and therefore more reproducibility and predictability. However, they have the disadvantages that they (1) offer a low, inadequate number of attachment sites per backbone, and (2) have a limited human-use experience. Similar disadvantages characterize the use of human serum albumin.
In light of the foregoing, an inexpensive and nontoxic molecular backbone delivery vehicle replete with high density molecular attachment capabilities is sorely needed.
It is an object of the invention to remedy or ameliorate one or more of the above-noted problems in the field and to provide useful alternatives.
Specifically, it is an object of the invention to provide a new or improved macromolecule delivery system for agents of any type, diagnostic, therapeutic, or otherwise.
It is a further object of the invention to provide a relatively nontoxic macromolecule possessing a high density of attachment sites relative to what has previously been described in the art.
It is a further object of the invention to improve delivery success and reduce administration volumes in techniques employing macromolecular backbones as delivery vehicles.
It is yet a further object of the invention to provide a macromolecule that is highly available, relatively inexpensive, and of demonstrable value in scientific research and medicine.
It is a further object of the invention to describe the chemical synthesis and product of new chemical attachment leashes having suitable flexibility.
In accordance with one or more of these objectives, in a first aspect the invention features a carrier molecule comprising a backbone, the backbone having affixed thereto a plurality of leash groups having structure xe2x80x94O(CH2)3S(CH2)2NH2.
In preferred embodiments, the backbone is derived from a polysaccharide, preferably dextran, and the leash structures are accomplished through reaction of an allyl group with aminoethanethiol. When dextran is used, it may be selected from any molecular weight appropriate for the ultimate use of the molecule, its leashes, and conjugates. Different diagnostic and therapeutic applications will call for different MW dextrans, as the person of ordinary skill in the art is aware.
In most advantageous embodiments, it is preferred that at least one chemical group be conjugated to the backbone via the amino groups of the leashes. These chemical groups may be selected from any of a variety of compounds having useful therapeutic or diagnostic uses, including but not limited to: chelators, receptor ligands, lectins, enzymatic substrates, nucleic acids, peptides, polysaccharides, monosaccharides, radiosensitizers, radioprotectors, and dyes. The groups need not be directly useful, but may be indirectly useful by permitting targeting to a given cell or tissue type such that another functional moiety attached to the backbone may perform the affirmative or negative function desired.
The high load and density of leashes per nontoxic backbone is an important aspect of the invention due to the significant kinetic advantages that are obtained for attachment and delivery. Relatively more therapeutic or diagnostic molecules may be delivered to work their purpose. This may include the very simplistic case in which ligands of high density may be simply attached to the backbone molecule such that they more effectively block certain receptors when administered or contacted thereto or therewith. Receptors impart cellular biochemical function. Blockage of that function may have a useful therapeutic value for a given indication and context. Thus, antagonists capable of competitive or noncompetive inhibition with normal or abnormal biological agents are contemplated.
Agonists can also be used to obtain the desired effect in that they may signal or stimulate endocytosis of the backbone moiety and agents to which they are attached, or can signal an intracellular cascade. Those of skill in the art will recognize the broad range of applications and implications for the many embodiments of the invention.
In especially preferred embodiments, the backbone carries both a ligand having a specific affinity for a given tissue or cell type, and a chelator molecule. The chelator normally has nitrogen groups possessing free electrons that adhere tightly to positively charged metal atoms and ions. Preferred chelators for use with the invention include DOTA, MAG3, and DTPA. DOTA is especially preferred because its geometry conveniently and tightly accommodates the gadolinium atom, which can be used for both Computed Tomography (CT) and Magnetic Resonance Imaging (MRI). The MAG3 chelator is preferred for complexation with the radioelement Technetium-99m (Tc-99m), which has a relatively short half-life of 6 hours. This half-life is compatible with biologic processes and clinical protocols, which for sentinel node detection is of sufficient duration to permit a surgeon to evaluate the precise location and extent of, e.g., tumor growth or metastases and remove it. This particular combination thus has excellent application in nuclear medicine.
In other, not necessarily mutually exclusive embodiments, the chelator may be combined with a non-radioisotope or element, e.g., an absorbing element (high density), or paramagnetic atom, each having special application, respectively, in computed tomography and nuclear magnetic or magnetic resonance imaging procedures. These are diagnostic procedures that permit imaging of various physiological or anatomical structures using characteristics of the high density or paramagnetic atoms introduced. Such diagnostic procedures may or may not be used in prelude to therapeutic procedures.
It may further be possible, using embodiments of the invention, to perform both diagnostic and therapeutic procedures at once, e.g., where some of the backbone leashes are conjugated with a diagnosing agent and others are conjugated with a therapeutic agent.
With respect to sentinel node detection, this particular diagnostic procedure takes advantage of high stability, nontoxicity, and high load and affinity supplied by the invention to improve the differentiation, isolation, and excision. This procedure will usually employ a chelator attached to a leash of the invention, to which can be further bound a radioatom such as technetium-99m. Other possibilities for the invention include the use of gadolinium, dysprosium, ytterbium, indium, and other elements possessing useful properties.
To target the above requires the additional use and employment of a receptor substrate, e.g., mannose, galactose, peptides, etc. Use of the term substrate does not necessarily suggest the ability to be acted upon by an enzyme, but can, and preferably does, refer to simple ligand:receptor binding. Thus the term xe2x80x9cligandxe2x80x9d may take many forms and constitutions, and may be used synonymously with xe2x80x9creceptor substratexe2x80x9d as used herein. Selecting a desired ligand or receptor substrate to target a given receptor is well within the level of skill in the art. Literally thousands of interactions are known which can be exploited for a given application. Furthermore, the targeted receptor may be viral, bacterial, protozoan, fungal, plant, insect, and not necessarily animal or mammalian in origin.
In terms of the degree of allylation, leash attachment, and conjugation, it may be anywhere from 1 to 100%, with 100% preferred. However, where not all leashes are ultimately conjugated, other embodiments may contemplate the addition of a noncharged xe2x80x9cblockingxe2x80x9d molecule, e.g., a methyl, alkyl, or aryl group to the free amino terminus or termini such that the overall molecule is less charged. In many applications, the less charge the better, as the person of skill is aware.
In a second aspect, the invention features, in addition to the products and applications of the first aspect, methods of producing substantially cross-link-free carrier molecules having a plurality of amino terminated leashes. By xe2x80x9csubstantially cross-link-freexe2x80x9d is meant cross-links introduced by man between molecules of the same type, e.g., glucose units in the same dextran molecule. The term is not meant to embrace the specific, intended conjugation of heteromolecules as described herein to the leashes of the backbone. It is further recognized that backbones, e.g., polysaccharides, may be naturally cross-linked to begin with; the term xe2x80x9csubstantially cross-link-freexe2x80x9d specifically excludes such phenomena.
The term xe2x80x9csubstantiallyxe2x80x9d is used to acknowledge that certain minimal, artifactual cross-linkages may nevertheless still occur, e.g., through the presence of contaminants or the use of sub-optimum attachment conditions, but that such limited cross-linking is tolerable. In this regard, the term is used to denote that a great object of the invention (to thwart, control or minimize man-made cross-linking phenomena associated with the introduction of conventional leashes to a backbone) is not significantly compromised. As noted, conventional procedures were flawed in this regard, and the invention significantly rectifies this through the use, e.g., of bifunctional groups such as in the preferred embodiment where aminoethanethiol is used. Prior to the invention, it was difficult if not impossible to control these unintended xe2x80x9cside reactionsxe2x80x9d, the result of which was to increase molecular weight above, and lower solubility below, tolerable and useful limits.
A preferred method embodiment preferably comprises providing a backbone molecule having a plurality of hydroxyl groups, allylating at least a portion of said hydroxyl groups on said backbone molecule to produce an allyl derivative of said backbone, reacting said allyl groups of said allyl derivative with a compound comprising an amino terminus and a second terminus, said second terminus specifically reactive with said allyl groups of said allyl derivative; and reacting said allyl derivative with said compound to produce a substantially cross-link-free carrier molecule having a plurality of amino terminated leashes.
In preferred embodiments, the backbone is, once again, a polysaccharide, preferably dextran. Preferably, although not necessarily, the compound used to create and affix the leashes to the allyl derivative backbone molecule is aminoethanethiol.
In further method embodiments, conjugation is contemplated as described for the first aspect, e.g., using at least one member selected from the group consisting of chelators, receptor ligands, enzymatic substrates, nucleic acids, peptides, polysaccharides, monosaccharides, radiosensitizers, radioprotectors, and dyes. The dyes can be fluorescent or otherwise distinguishable using visible and/or assistance means common in the art.
Other method embodiments track those already noted for the first aspect.
Still further aspects of the invention are more specific products and product-by-processes. For example, an MRI agent synthesized from the molecule of the first aspect is claimed, as is an MRI agent made according to the methods of the second aspect. Also claimed is a CT agent synthesized from or according to any of the first two aspects. Finally, sentinel node imaging agents are claimed that feature or employ the first or second aspects, and any feasible combination of those aspects"" specific embodiments.